This invention relates to a spectrophotometric assay for the detection of acetaminophen in aqueous fluids which is carried out with a dry analytical element.
Acetaminophen is a widely used analgesic. It is available without a prescription and is often used when aspirin may present problems to a patient. At therapeutic doses, serum concentration is usually below 50 mg/L. Toxicity is usually observed if the serum concentration four hours after ingestion of the drug is greater than 300 mg/L. One effect of overdose is liver toxicity. The need for an accurate method of determining the concentration of acetaminophen in serum is therefore apparent.
Known methods of assaying anilides such as acetaminophen utilize arylacylamidase (E.C.3.5.1.13) and an oxidizing agent. Arylacylamidase cleaves the amide bond of the anilide to produce acetate and an aniline such as p-aminophenol. The aniline is then made to react with a color-forming compound like phenol in the presence of an oxidizing agent such as permanganate or the metal salts of copper or iron to form a color compound like indophenol which can be detected at 615 nm. Other methods use oxidizing agents like periodate or persulfates. U.S. Pat. Nos. 4,999,288 and 4,430,433 are typical.
One disadvantage of these methods is that most metal salts oxidize the aniline very slowly unless they are at alkaline pH, a condition that inactivates arylacylamidase.
U.S. Pat. No. 4,675,290 (issued Jun. 23, 1987, to Matsumoto) discloses a method of assaying peptidase to enzyme activity which comprises treating a synthetic dibrominated amide (as substrate) with a sample containing peptidase thereby liberating a dibrominated aniline; oxidizing the thus-liberated dibrominated aniline with an oxidase (e.g. ascorbate oxidase) which consumes oxygen and forms pigment by oxidative condensation of said aniline in the presence of a coupler of a defined formula. This reaction takes place in solution.
Matsumoto""s method uses an enzyme and might therefore overcome the problems encountered with the inorganic oxidizing agents of the prior art. However, Matsumoto employs a synthetic dibrominated substrate and is therefore not directly applicable to a test for acetaminophen. There is evidence in the art that in the case of oxidative coupling of phenols for color formation, color yield can be increased when certain halogenophenols are used in the color-forming reaction. However acetaminophen as it appears in biological fluids to be tested is not halogenated and is therefore chemically distinguishable from the dibrominated synthetic substrate used in Matsumoto""s method.
Further, Matsumoto disclosed a solution assay. An alternative and more convenient assay involves xe2x80x9cdryxe2x80x9d chemistry, a term that refers to methods and techniques that are carried out using chemical reagents contained in various xe2x80x9cdry-to-the-touchxe2x80x9d test elements such as xe2x80x9cdip and readxe2x80x9d test strips, multilayer test elements and the like. xe2x80x9cDryxe2x80x9d methods require no liquid for reconstitution or analysis other than the test sample.
Reagents employed in solution assays often do not perform well when adapted to a dry format. Dry elements utilize minute amounts of reagents and test samples and so in a dry element for acetaminophen assay, it is important to employ reagents that give a clear and precise color signal when aniline is oxidatively coupled to a coupler to provide color.
The range of compounds that could be chosen as potential couplers is very wide. Matsumoto neither suggests nor gives guidance to those color forming oxidative couplers that would perform well in dry chemistry. The problem is that the selection of a suitable oxidative coupler for a dry element is essentially empirical.
It would be desirable to have a dry assay for determining acetaminophen in biological fluids comprising enzymes and other reagents which remain active when combined and coated in dry format and which give a clear and precise signal on oxidative coupling with a suitable coupler.
The present invention selects for use a coupling agent that provides a detectable color change and precise determination of acetaminophen in a dry format. It was discovered that a water-soluble coupling agent was required and that a water-insoluble coupling agent, even if appropriately dispersed, did not work well.
The element of the present invention avoids the inorganic oxidizing gents taught by the prior art and uses instead mild oxidizing agents, such as an enzyme or a ferricyanide. In dry analytical elements, oxidizing agents like Cu++ and most Fe+++ salts are known to attack a gelatin matrix by crosslinking and thus hardening the gelatin. The use of an enzyme as an oxidizing agent overcomes this problem. Suitable oxidizing enzymes include ascorbic acid oxidase, lactase and tyrosinase.
The ferricyanide is not as strong an oxidizing agent as most Fe+++ salts or other metal oxidizing agents (see Table A). It is unexpected, therefore, that a ferricyanide would be able to provide fast reaction kinetics at neutral pHs without it damaging the dry element structure. Suitable ferricyanides include the alkali metal and alkaline earth metal ferricyanides such as, for example, potassium, sodium and calcium ferricyanide. The preferred ferricyanides are the alkali metal ferricyanides.
Accordingly, there is provided an analytical element for the determination of acetaminophen in an aqueous fluid comprising a support having thereon at least one reagent layer and containing in said reagent layer:
(a) an arylacylamidase enzyme;
(b) an oxidizing enzyme or a ferricyanide capable of oxidatively coupling paraaminophenol to a coupling agent to form a color compound.
(c) a water-soluble, color-forming, coupling agent of the general structure: 
wherein
R is a water-solubilizing group selected from xe2x80x94(CH2)nX where n is 1 to 5 and X is xe2x80x94SO3M where M is hydrogen, an alkali metal, an alkaline earth metal or an ammonium (NH4+) cation, or xe2x80x94N(R7)3+Zxe2x80x94 where each R7 is independently selected from alkyl of 1 to 4 carbon atoms such as methyl, ethyl, propyl and butyl, and Z is an acid anion such as chloride, bromide, iodide, fluoride, p-toluenesulfonate, and the like, or X is (xe2x80x94OCH2CH2)yOH where y is 2 to 5;
R1 and R6 are taken together to represent an ethylene, trimethylene, or tetramethylene group which forms a partially saturated ring;
R2, R3, and R4 are independently selected from hydrogen, alkyl of 1 to 4 carbon atoms, and alkoxy of 1 to 4 carbon atoms.
Another embodiment of the invention provides a method for determining acetaminophen in an aqueous liquid comprising the steps of:
a. contacting a sample of the aqueous liquid with the analytical element described above; and
b. correlating the amount of color compound formed to the concentration of acetaminophen in the fluid.
An advantage of the invention is that the water-soluble coupling agent and either the enzyme-catalyzed oxidative coupling or the ferricyanide coupling allow for a rapid reaction. We were able to develop a detectable color signal in 57 seconds. Water-insoluble coupling agents involve much slower reactions.
It was surprising to find that in order to obtain an acceptable amount of color product in the desired time (5 minutes), it was necessary to combine a water-soluble coupling agent and an enzyme. Even in solution assays, in order to obtain results in 5 minutes, the oxidizing enzyme (e.g. ascorbate oxidase) had to be premixed with a water-soluble coupling agent prior to dilution with buffer. The reason for this is not known. While not wishing to be bound by scientific theories, Applicants believe that water solubility of the coupling agent allows it to mix sufficiently with the enzyme in the analytical element during storage (i.e. between manufacture and use). It appears that prior contact between the coupling agent and the oxidizing enzyme before reaction is necessary to obtain a satisfactory color reaction in 5 minutes.